The invention concerns an apparatus for qualitative and/or quantitative detection of analytes, particularly in biological samples, by means of receptor ligand binding using a magnetizing device for the production of a magnetic field at a location of the sample and having a detection device for measurement of magnetic properties of the sample.
Similar apparatus can be inferred from prior art through the description of a measurement procedure for execution on an appropriate apparatus in accordance with JP 63090765-A2.
Apparatus for the measurement of receptor ligand binding are based on the measurement of signals generated by signal producing labels with which structure-specific substances are marked. The most sensitive current apparatus are based on the detection of radioactive marking substances (Radio Immuno Assay, RIA). The utilization of radioactive labels has obvious disadvantages, for example problems with storage and disposal of the radioactive substances. In addition, this type of apparatus requires a device for separating the bound from the unbound labels. Without this separation, a quantitative prediction is generally not possible.
Alternative apparatus are based on the optical measurement of agglutination, fluorescence, and color reactions. (FIA, ELISA). Herein, one is dealing substantially with photodetectors. Separation is also normally required in these methods for quantitative analysis. On the other hand there are a large number of apparatus for the determination of magnetic properties of samples which, however, have generally not been applied to the direct detection of receptor ligand binding up to this point in time.
A number of apparatus are known in the art which are based on methods using magnetic labels. For example, application of a magnetic field causes motion of magnetically marked particles which can be observed using, for example, a laser. In addition, there are apparatus based on methods with which magnetic labels are utilized for separation of bound and unbound components. In contrast thereto, there are very few apparatus which are based on measurement of the magnetic properties of the sample.
JP 63090765-A2 describes a SQUID immuno assay method based on magnetically marked antibodies or antigens. Unbound portions must, however, be removed from the sample (separation) following the antibody-antigen reaction. An appropriate apparatus therefor must consequently contain a device for separating the bound from the unbound labels. The magnetization of the sample is measured subsequent to separation in the presence of a magnetic field, i.e. measurement of the magnetization takes place in the field.
U.S. Pat. No. 4,913,883 describes an apparatus for immuno-agglutination assay. Same is based on the measurement of the agglutination of antibodies marked with magnetic particles in the .mu.m size range. The apparatus necessarily comprises a device for isolating the agglutinate and a device for transport of this agglutinate through the detection device using a fluid stream.
JP 3-220442 A discloses a measuring process for carrying out agglutination immuno-assays with which the amount of agglutination is antibody determined using a method for measurement of the particle size of agglomerated magnetic particles disclosed in the publication. The method consists of switching a magnetic field which penetrates the stationary fluid sample and measuring the residual magnetic flux density of the agglomerated magnetic particles.
In accordance with JP 3-220442 A determination of the degree of agglutination can also be carried out by means of optical processes for determination of particle sizes. Towards this end the published process solely concerns a method for the determination of the particle size of magnetic particles resulting from agglutination which can only be utilized in the manner described for agglomerates of magnetic particles, wherein the particle size of the agglomerate lies in the micrometer range.
A measuring process described in U.S. Pat. No. 5,486,457 serves for the determination of the mobility of magnetic particles bound to cells. The apparatus described therein measures magnetic fields in the presence of a weaker magnetic field which is rotated through 90.degree. with respect to the magnetizing field.
An article by Valbeg et al. in Science 1984, volume 424, pp. 513-516 discloses a method based on magnetic field measurements under rotation of magnetic particles with particle sizes of typically 0.7 .mu.m. Lock-in-technology is explicitly used to increase the measuring sensitivity. This is a modulation process with which the measured signal is recorded in a narrow band fashion.
The article by Philo et al. in Rev. Sci. Instrum. 1977, Volume 48, pp. 1529-1536 describes a process with which volume susceptibilities can be measured with the assistance of SQUID technology. Those advantages of SQUIDs for high-sensitivity measurements which could be useful to future instrumentation are explicitly mentioned.
The German patent applications DE 195 03 664.6 and DE 195 08 772.0, which do not constitute prior art, describe methods and compounds for the magnetorelaxometric detection of analytes or for the detection of analytes by means of remanence measurements. In the following, magnetorelaxometric detection designates the binding-specific detection of analytes in liquid or solid phase characterized by the use of ferro- or ferrimagnetic colloidal particles as a detectable magnetic marking for the detection of analytes by means of ligand receptor binding with relaxation of their magnetization being determined as the measured quantity. Below, detection of analytes by means of remanence measurement (also referred to below as measurements of the binding remanence) designates the binding-specific detection of analytes in liquid or solid phases characterized by the use of stable or quasi-stable ferro- or ferrimagnetic substances as detectable magnetic markings for the detection of analytes by means of ligand receptor binding with the remanence of their magnetization being determined as the measured quantity. In the latter two methods:
I) the relaxation (the time decay of the magnetization) of the sample is measured directly after switch-off or removal of the magnetic field or PA1 II) the frequency-dependent magnetization of the sample in the presence of a magnetizing field is measured or PA1 III) the binding-specific remanence of the sample is measured following magnetization. PA1 1. sufficiently suppress external interfering signals (for example power line hum, fluctuations in the earth's magnetic field) and to avoid production of internal interfering signals to the extent possible, PA1 2. avoid difficult or expensive magnetic shielding, as well as PA1 3. effect simple and rapid sample change.
Towards this end it is desirable to:
A new type of apparatus is therefore needed to carry out the above mentioned new methods which facilitates highly sensitive quantitative and qualitative detection of receptor ligand binding through measurement of the magnetic properties of samples.
Since no apparatus of this type are currently known in the art which are suitable for a routine economical execution of the above described new procedures, it is the purpose of the present invention to introduce apparatus of the above mentioned kind with which these new types of measurements can be carried out.
In accordance with a first aspect of the present invention this purpose is achieved in that, with an apparatus having the above mentioned features, the magnetizing device is spatially disposed with respect to the detection device in such a fashion that the magnetic field produced by the magnetizing device at the location of the magnetization is attenuated by at least a factor of 10, preferentially by a factor of 1000 or more at the location occupied by the sample during the measurement.